Novel human ion-exchanger proteins and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. Provisional Application No. 60/235,745, which was filed on Sep. 27, 2000 and is herein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the discovery, identification, and characterization of novel human polynucleotides encoding proteins that share sequence similarity with mammalian membrane proteins. The invention encompasses the described polynucleotides, host cell expression systems, the encoded proteins, fusion proteins, polypeptides and peptides, antibodies to the encoded proteins and peptides, and genetically engineered animals that either lack or over express the disclosed genes, antagonists and agonists of the proteins, and other compounds that modulate the expression or activity of the proteins encoded by the disclosed genes that can be used for diagnosis, drug screening, clinical trial monitoring, the treatment of diseases and disorders, and cosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

[0003] Membrane proteins can serve as recognition markers, mediate signal transduction, and can mediate or facilitate the passage of materials across the lipid bilayer. As such, membrane proteins, are proven drug targets.

3. SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification, and characterization of nucleotides that encode novel human proteins and the corresponding amino acid sequences of these proteins. The novel human proteins (NHPs) described for the first time herein share structural similarity with mammalian sodium-calcium exchanger proteins, sodium-calcium potassium exchanger proteins, and potassium dependent versions of the same.

[0005] The novel human nucleic acid sequences described herein encode alternative proteins/open reading frames (ORFs) of 603, 316 and 353 amino acids in length (SEQ ID NOS: 2, 4, and 6).

[0006] The invention also encompasses agonists and antagonists of the described NHPs, including small molecules, large molecules, mutant NHPs, or portions thereof, that compete with native NHP, peptides, and antibodies, as well as nucleotide sequences that can be used to inhibit the expression of the described NHPs (e.g., antisense and ribozyme molecules, and open reading frame or regulatory sequence replacement constructs) or to enhance the expression of the described NHPs (e.g., expression constructs that place the described polynucleotide under the control of a strong promoter system), and transgenic animals that express a NHP sequence, or “knock-outs” (which can be conditional) that do not express a functional NHP. Knock-out mice can be produced in several ways, one of which involves the use of mouse embryonic stem cells (“ES cells”) lines that contain gene trap mutations in a murine homolog of at least one of the described NHPs. When the unique NHP sequences described in SEQ ID NOS:1-7 are “knocked-out” they provide a method of identifying phenotypic expression of the particular gene as well as a method of assigning function to previously unknown genes. In addition, animals in which the unique NHP sequences described in SEQ ID NOS:1-7 are “knocked-out” provide a unique source in which to elicit antibodies to homologous and orthologous proteins that would have been previously viewed by the immune system as “self” and therefore would have failed to elicit significant antibody responses. To these ends, gene trapped knockout ES cells have been generated in murine homologs of the described NHPs.

[0007] Additionally, the unique NHP sequences described in SEQ ID NOS:1-7 are useful for the identification of protein coding sequence and mapping a unique gene to a particular chromosome. These sequences identify actual, biologically relevant, exon splice junctions as opposed to those that might have been predicted bioinformatically from genomic sequence alone. The sequences of the present invention are also useful as additional DNA markers for restriction fragment length polymorphism (RFLP) analysis, and in forensic biology.

[0008] Further, the present invention also relates to processes for identifying compounds that modulate, i.e., act as agonists or antagonists, of NHP expression and/or NHP activity that utilize purified preparations of the described NHPs and/or NHP product, or cells expressing the same. Such compounds can be used as therapeutic agents for the treatment of any of a wide variety of symptoms associated with biological disorders or imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides the sequences of the NHP ORFs encoding the described NHP amino acid sequences. SEQ ID NO:7 describes a polynucleotide encoding a NHP ORF with regions of flanking sequence.

5. DETAILED DESCRIPTION OF THE INVENTION

[0010] The NHPs described for the first time herein are novel proteins that may be expressed in, inter alia, human cell lines, fetal brain, brain, pituitary, cerebellum, spinal cord, thymus, spleen, bone marrow, lymph node, trachea, lung, kidney, fetal liver, liver, prostate, testis, thyroid, adrenal gland, pancreas, salivary gland, stomach, small intestine, colon, skeletal muscle, heart, uterus, placenta, mammary gland, adipose, skin, esophagus, bladder, cervix, rectum, pericardium, hypothalamus, ovary, fetal kidney, fetal lung, gall bladder, tongue, 6-, 9-, and 12 week embryo, adenocarcinoma, and embryonic carcinoma cells.

[0011] The present invention encompasses the nucleotides presented in the Sequence Listing, host cells expressing such nucleotides, the expression products of such nucleotides, and: (a) nucleotides that encode mammalian homologs of the described genes, including the specifically described NHPs, and the NHP products; (b) nucleotides that encode one or more portions of the NHPs that correspond to functional domains, and the polypeptide products specified by such nucleotide sequences, including but not limited to the novel regions of any active domain(s); (c) isolated nucleotides that encode mutant versions, engineered or naturally occurring, of the described NHPs in which all or a part of at least one domain is deleted or altered, and the polypeptide products specified by such nucleotide sequences, including but not limited to soluble proteins and peptides in which all or a portion of the signal (or hydrophobic transmembrane) sequence is deleted; (d) nucleotides that encode chimeric fusion proteins containing all or a portion of a coding region of an NHP, or one of its domains (e.g., a receptor or ligand binding domain, accessory protein/self-association domain, etc.) fused to another peptide or polypeptide; or (e) therapeutic or diagnostic derivatives of the described polynucleotides such as oligonucleotides, antisense polynucleotides, ribozymes, dsRNA, or gene therapy constructs comprising a sequence first disclosed in the Sequence Listing.

[0012] As discussed above, the present invention includes: (a) the human DNA sequences presented in the Sequence Listing (and vectors comprising the same) and additionally contemplates any nucleotide sequence encoding a contiguous NHP open reading frame (ORF) that hybridizes to a complement of a DNA sequence presented in the Sequence Listing under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes a functionally equivalent expression product. Additionally contemplated are any nucleotide sequences that hybridize to the complement of a DNA sequence that encodes and expresses an amino acid sequence presented in the Sequence Listing under moderately stringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yet still encodes a functionally equivalent NHP product. Functional equivalents of a NHP include naturally occurring NHPs present in other species and mutant NHPs whether naturally occurring or engineered (by site directed mutagenesis, gene shuffling, directed evolution as described in, for example, U.S. Pat. No. 5,837,458). The invention also includes degenerate nucleic acid variants of the disclosed NHP polynucleotide sequences.

[0013] Additionally contemplated are polynucleotides encoding NHP ORFs, or their functional equivalents, encoded by polynucleotide sequences that are about 99, 95, 90, or about 85 percent similar or identical to corresponding regions of the nucleotide sequences of the Sequence Listing (as measured by BLAST sequence comparison analysis using, for example, the GCG sequence analysis package using standard default settings).

[0014] The invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the described NHP gene nucleotide sequences. Such hybridization conditions may be highly stringent or less highly stringent, as described above. In instances where the nucleic acid molecules are deoxyoligonucleotides (“DNA oligos”), such molecules are generally about 16 to about 100 bases long, or about 20 to about 80, or about 34 to about 45 bases long, or any variation or combination of sizes represented therein that incorporate a contiguous region of sequence first disclosed in the Sequence Listing. Such oligonucleotides can be used in conjunction with the polymerase chain reaction (PCR) to screen libraries, isolate clones, and prepare cloning and sequencing templates, etc.

[0015] Alternatively, such NHP oligonucleotides can be used as hybridization probes for screening libraries, and assessing gene expression patterns (particularly using a micro array or high-throughput “chip” format). Additionally, a series of the described NHP oligonucleotide sequences, or the complements thereof, can be used to represent all or a portion of the described NHP sequences. An oligonucleotide or polynucleotide sequence first disclosed in at least a portion of one or more of the sequences of SEQ ID NOS: 1-7 can be used as a hybridization probe in conjunction with a solid support matrix/substrate (resins, beads, membranes, plastics, polymers, metal or metallized substrates, crystalline or polycrystalline substrates, etc.). Of particular note are spatially addressable arrays (i.e., gene chips, microtiter plates, etc.) of oligonucleotides and polynucleotides, or corresponding oligopeptides and polypeptides, wherein at least one of the biopolymers present on the spatially addressable array comprises an oligonucleotide or polynucleotide sequence first disclosed in at least one of the sequences of SEQ ID NOS: 1-7, or an amino acid sequence encoded thereby. Methods for attaching biopolymers to, or synthesizing biopolymers on, solid support matrices, and conducting binding studies thereon are disclosed in, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405, the disclosures of which are herein incorporated by reference in their entirety.

[0016] Addressable arrays comprising sequences first disclosed in SEQ ID NOS:1-7 can be used to identify and characterize the temporal and tissue specific expression of a gene. These addressable arrays incorporate oligonucleotide sequences of sufficient length to confer the required specificity, yet be within the limitations of the production technology. The length of these probes is within a range of between about 8 to about 2000 nucleotides. Preferably the probes consist of 60 nucleotides and more preferably 25 nucleotides from the sequences first disclosed in SEQ ID NOS:1-7.

[0017] For example, a series of the described oligonucleotide sequences, or the complements thereof, can be used in chip format to represent all or a portion of the described sequences. The oligonucleotides, typically between about 16 to about 40 (or any whole number within the stated range) nucleotides in length can partially overlap each other and/or the sequence may be represented using oligonucleotides that do not overlap. Accordingly, the described polynucleotide sequences shall typically comprise at least about two or three distinct oligonucleotide sequences of at least about 8 nucleotides in length that are each first disclosed in the described Sequence Listing. Such oligonucleotide sequences can begin at any nucleotide present within a sequence in the Sequence Listing and proceed in either a sense (5′-to-3′) orientation vis-a-vis the described sequence or in an antisense orientation.

[0018] Microarray-based analysis allows the discovery of broad patterns of genetic activity, providing new understanding of gene functions and generating novel and unexpected insight into transcriptional processes and biological mechanisms. The use of addressable arrays comprising sequences first disclosed in SEQ ID NOS:1-7 provides detailed information about transcriptional changes involved in a specific pathway, potentially leading to the identification of novel components or gene functions that manifest themselves as novel phenotypes.

[0019] Probes consisting of sequences first disclosed in SEQ ID NOS:1-7 can also be used in the identification, selection and validation of novel molecular targets for drug discovery. The use of these unique sequences permits the direct confirmation of drug targets and recognition of drug dependent changes in gene expression that are modulated through pathways distinct from the drugs intended target. These unique sequences therefore also have utility in defining and monitoring both drug action and toxicity.

[0020] As an example of utility, the sequences first disclosed in SEQ ID NOS:1-7 can be utilized in microarrays or other assay formats, to screen collections of genetic material from patients who have a particular medical condition. These investigations can also be carried out using the sequences first disclosed in SEQ ID NOS:1-7 in silico and by comparing previously collected genetic databases and the disclosed sequences using computer software known to those in the art.

[0021] Thus the sequences first disclosed in SEQ ID NOS:1-7 can be used to identify mutations associated with a particular disease and also as a diagnostic or prognostic assay.

[0022] Although the presently described sequences have been specifically described using nucleotide sequence, it should be appreciated that each of the sequences can uniquely be described using any of a wide variety of additional structural attributes, or combinations thereof. For example, a given sequence can be described by the net composition of the nucleotides present within a given region of the sequence in conjunction with the presence of one or more specific oligonucleotide sequence(s) first disclosed in the SEQ ID NOS: 1-7. Alternatively, a restriction map specifying the relative positions of restriction endonuclease digestion sites, or various palindromic or other specific oligonucleotide sequences can be used to structurally describe a given sequence. Such restriction maps, which are typically generated by widely available computer programs (e.g., the University of Wisconsin GCG sequence analysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich., etc.), can optionally be used in conjunction with one or more discrete nucleotide sequence(s) present in the sequence that can be described by the relative position of the sequence relative to one or more additional sequence(s) or one or more restriction sites present in the disclosed sequence. For oligonucleotide probes, highly stringent conditions may refer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos). These nucleic acid molecules may encode or act as NHP gene antisense molecules, useful, for example, in NHP gene regulation (for and/or as antisense primers in amplification reactions of NHP gene nucleic acid sequences). With respect to NHP gene regulation, such techniques can be used to regulate biological functions. Further, such sequences may be used as part of ribozyme and/or triple helix sequences that are also useful for NHP gene regulation.

[0023] Inhibitory antisense or double stranded oligonucleotides can additionally comprise at least one modified base moiety that is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[0024] The antisense oligonucleotide can also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0025] In yet another embodiment, the antisense oligonucleotide will comprise at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

[0026] In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330). Alternatively, double stranded RNA can be used to disrupt the expression and function of a targeted NHP.

[0027] Oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.

[0028] Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual (and periodic updates thereof), Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.

[0029] Alternatively, suitably labeled NHP nucleotide probes can be used to screen a human genomic library using appropriately stringent conditions or by PCR. The identification and characterization of human genomic clones is helpful for identifying polymorphisms (including, but not limited to, nucleotide repeats, microsatellite alleles, single nucleotide polymorphisms, or coding single nucleotide polymorphisms), determining the genomic structure of a given locus/allele, and designing diagnostic tests. For example, sequences derived from regions adjacent to the intron/exon boundaries of the human gene can be used to design primers for use in amplification assays to detect mutations within the exons, introns, splice sites (e.g., splice acceptor and/or donor sites), etc., that can be used in diagnostics and pharmacogenomics.

[0030] For example, the present sequences can be used in restriction fragment length polymorphism (RFLP) analysis to identify specific individuals. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification (as generally described in U.S. Pat. No. 5,272,057, incorporated herein by reference). In addition, the sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e., another DNA sequence that is unique to a particular individual). Actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.

[0031] Further, a NHP gene homolog can be isolated from nucleic acid from an organism of interest by performing PCR using two degenerate or “wobble” oligonucleotide primer pools designed on the basis of amino acid sequences within the NHP products disclosed herein. The template for the reaction may be total RNA, mRNA, and/or cDNA obtained by reverse transcription of mRNA prepared from human or non-human cell lines or tissue known or suspected to express an allele of a NHP gene. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequence of the desired NHP gene. The PCR fragment can then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment can be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment can be used to isolate genomic clones via the screening of a genomic library.

[0032] PCR technology can also be used to isolate full length cDNA sequences. For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source (i.e., one known, or suspected, to express a NHP gene). A reverse transcription (RT) reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5′ end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be “tailed” using a standard terminal transferase reaction, the hybrid may be digested with RNase H, and second strand synthesis may then be primed with a complementary primer. Thus, cDNA sequences upstream of the amplified fragment can be isolated. For a review of cloning strategies that can be used, see e.g., Sambrook et al., 1989, supra.

[0033] A cDNA encoding a mutant NHP sequence can be isolated, for example, by using PCR. In this case, the first cDNA strand may be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual putatively carrying a mutant NHP allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5′ end of the normal sequence. Using these two primers, the product is then amplified via PCR, optionally cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant NHP allele to that of a corresponding normal NHP allele, the mutation(s) responsible for the loss or alteration of function of the mutant NHP gene product can be ascertained.

[0034] Alternatively, a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry a mutant NHP allele (e.g., a person manifesting a NHP-associated phenotype such as, for example, osteoporosis, obesity, high blood pressure, connective tissue disorders, infertility, etc.), or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express a mutant NHP allele. A normal NHP gene, or any suitable fragment thereof, can then be labeled and used as a probe to identify the corresponding mutant NHP allele in such libraries. Clones containing mutant NHP sequences can then be purified and subjected to sequence analysis according to methods well known to those skilled in the art.

[0035] Additionally, an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant NHP allele in an individual suspected of or known to carry such a mutant allele. In this manner, gene products made by the putatively mutant tissue can be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against a normal NHP product, as described below. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold Spring Harbor.) Additionally, screening can be accomplished by screening with labeled NHP fusion proteins, such as, for example, alkaline phosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In cases where a NHP mutation results in an expression product with altered function (e.g., as a result of a missense or a frameshift mutation), polyclonal antibodies to NHP are likely to cross-react with a corresponding mutant NHP expression product. Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods well known in the art.

[0036] The invention also encompasses (a) DNA vectors that contain any of the foregoing NHP coding sequences and/or their complements (i.e., antisense); (b) DNA expression vectors that contain any of the foregoing NHP coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences (for example, baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporated by reference); (c) genetically engineered host cells that contain any of the foregoing NHP coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell; and (d) genetically engineered host cells that express an endogenous NHP sequence under the control of an exogenously introduced regulatory element (i.e., gene activation). As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include but are not limited to the cytomegalovirus (hCMV) immediate early gene, regulatable, viral elements (particularly retroviral LTR promoters), the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase (PGK), the promoters of acid phosphatase, and the promoters of the yeast α-mating factors.

[0037] The present invention also encompasses antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists and agonists of a NHP, as well as compounds or nucleotide constructs that inhibit expression of a NHP sequence (transcription factor inhibitors, antisense and ribozyme molecules, or open reading frame sequence or regulatory sequence replacement constructs), or promote the expression of a NHP (e.g., expression constructs in which NHP coding sequences are operatively associated with expression control elements such as promoters, promoter/enhancers, etc.).

[0038] The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences, antibodies, antagonists and agonists can be useful for the detection of mutant NHPs or inappropriately expressed NHPs for the diagnosis of disease. The NHP proteins or peptides, NHP fusion proteins, NHP nucleotide sequences, host cell expression systems, antibodies, antagonists, agonists and genetically engineered cells and animals can be used for screening for drugs (or high throughput screening of combinatorial libraries) effective in the treatment of the symptomatic or phenotypic manifestations of perturbing the normal function of NHP in the body. The use of engineered host cells and/or animals may offer an advantage in that such systems allow not only for the identification of compounds that bind to the endogenous receptor for an NHP, but can also identify compounds that trigger NHP-mediated activities or pathways.

[0039] Finally, the NHP products can be used as therapeutics. For example, soluble derivatives such as NHP peptides/domains corresponding to NHPs, NHP fusion protein products (especially NHP-Ig fusion proteins, i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHP antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists or agonists (including compounds that modulate or act on downstream targets in a NHP-mediated pathway) can be used to directly treat diseases or disorders. For instance, the administration of an effective amount of soluble NHP, or a NHP-IgFc fusion protein or an anti-idiotypic antibody (or its Fab) that mimics the NHP could activate or effectively antagonize the endogenous NHP receptor. Nucleotide constructs encoding such NHP products can be used to genetically engineer host cells to express such products in vivo; these genetically engineered cells function as “bioreactors” in the body delivering a continuous supply of a NHP, a NHP peptide, or a NHP fusion protein to the body. Nucleotide constructs encoding functional NHPs, mutant NHPs, as well as antisense and ribozyme molecules can also be used in “gene therapy” approaches for the modulation of NHP expression. Thus, the invention also encompasses pharmaceutical formulations and methods for treating biological disorders.

[0040] Various aspects of the invention are described in greater detail in the subsections below.

5.1 THE NHP SEQUENCES

[0041] The cDNA sequences and the corresponding deduced amino acid sequences of the described NHPs are presented in the Sequence Listing. The NHP nucleotides were obtained from clustered genomic sequence, ESTs, and cDNAs from brain and pituitary gland cDNA libraries (Edge Biosystems, Gaithersburg, Md.). The gene encoding the described NHPs is apparently encoded on human chromosome 14 (see GENBANK accession no. AL118559).

[0042] A number of polymorphism were identified during the sequencing of the NHPs including a C/G at the nucleotide position represented by, for example, position 1147 of SEQ ID NO: 1 (which can result in a pro or ala at corresponding amino acid (aa) position 383), a T/G at nucleotide position 1163 (which can result in an val or gly at aa position 388), and a T/G at position 1193 (which can result in a val or gly at aa position 398). The present invention contemplates sequences comprising any of the above polymorphisms, as well as any and all combinations and permutations of the above.

[0043] An additional application of the described novel human polynucleotide sequences is their use in the molecular mutagenesis/evolution of proteins that are at least partially encoded by the described novel sequences using, for example, polynucleotide shuffling or related methodologies. Such approaches are described in U.S. Pat. Nos. 5,830,721 and 5,837,458, which are herein incorporated by reference in their entirety.

[0044] NHP gene products can also be expressed in transgenic animals. Animals of any species, including, but not limited to, worms, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate NHP transgenic animals.

[0045] Any technique known in the art may be used to introduce a NHP transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which is incorporated by reference herein in its entirety.

[0046] The present invention provides for transgenic animals that carry the NHP transgene in all their cells, as well as animals that carry the transgene in some, but not all their cells, i.e., mosaic animals or somatic cell transgenic animals. The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

[0047] When it is desired that a NHP transgene be integrated into the chromosomal site of the endogenous NHP gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous NHP gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous NHP gene (i.e., “knockout” animals).

[0048] The transgene can also be selectively introduced into a particular cell type, thus inactivating the endogenous NHP gene in only that cell type, by following, for example, the teaching of Gu et al., 1994, Science, 265:103-106. The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

[0049] Once transgenic animals have been generated, the expression of the recombinant NHP gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques that include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of NHP gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the NHP transgene product.

5.2 NHPS AND NHP POLYPEPTIDES

[0050] NHPs, NHP polypeptides, NHP peptide fragments, mutated, truncated, or deleted forms of the NHPs, and/or NHP fusion proteins can be prepared for a variety of uses. These uses include, but are not limited to, the generation of antibodies, as reagents in diagnostic assays, for the identification of other cellular gene products related to a NHP, as reagents in assays for screening for compounds that can be used as pharmaceutical reagents useful in the therapeutic treatment of mental, biological, or medical disorders and disease. Given the similarity information and expression data, the described NHPs can be targeted (by drugs, oligos, antibodies, etc.,) in order to treat disease, or to therapeutically augment the efficacy of, for example, chemotherapeutic agents used in the treatment of cancer, arthritis, or as antiviral agents.

[0051] The Sequence Listing discloses the amino acid sequences encoded by the described NHP sequences. The NHPs display initiator methionines in DNA sequence contexts consistent with translation initiation sites, and a hydrophobic region near the N-terminus that may serve as a signal sequence, which indicates that the described NHPs can be secreted, membrane-associated, or cytoplasmic.

[0052] The NHP amino acid sequences of the invention include the amino acid sequence presented in the Sequence Listing as well as analogues and derivatives thereof. Further, corresponding NHP homologues from other species are encompassed by the invention. In fact, any NHP protein encoded by the NHP nucleotide sequences described above are within the scope of the invention as are any novel polynucleotide sequences encoding all or any novel portion of an amino acid sequence presented in the Sequence Listing. The degenerate nature of the genetic code is well known, and, accordingly, each amino acid presented in the Sequence Listing, is generically representative of the well known nucleic acid “triplet” codon, or in many cases codons, that can encode the amino acid. As such, as contemplated herein, the amino acid sequences presented in the Sequence Listing, when taken together with the genetic code (see, for example, Table 4-1 at page 109 of “Molecular Cell Biology”, 1986, J. Darnell et al. eds., Scientific American Books, New York, N.Y., herein incorporated by reference) are generically representative of all the various permutations and combinations of nucleic acid sequences that can encode such amino acid sequences.

[0053] The invention also encompasses proteins that are functionally equivalent to the NHPs encoded by the presently described nucleotide sequences as judged by any of a number of criteria, including, but not limited to, the ability to bind and cleave a substrate of a NHP, or the ability to effect an identical or complementary downstream pathway, or a change in cellular metabolism (e.g., proteolytic activity, ion flux, tyrosine phosphorylation, etc.). Such functionally equivalent NHP proteins include, but are not limited to, additions or substitutions of amino acid residues within the amino acid sequence encoded by the NHP nucleotide sequences described above, but that result in a silent change, thus producing a functionally equivalent expression product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0054] A variety of host-expression vector systems can be used to express the NHP nucleotide sequences of the invention. Where, as in the present instance, the NHP peptide or polypeptide is thought to be membrane protein, the hydrophobic regions of the protein can be excised and the resulting soluble peptide or polypeptide can be recovered from the culture media. Such expression systems also encompass engineered host cells that express a NHP, or functional equivalent, in situ. Purification or enrichment of a NHP from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such engineered host cells themselves may be used in situations where it is important not only to retain the structural and functional characteristics of the NHP, but to assess biological activity, e.g., in drug screening assays.

[0055] The expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing NHP nucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing NHP nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing NHP sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing NHP nucleotide sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0056] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the NHP product being expressed. For example, when a large quantity of such a protein is to be produced for the generation of pharmaceutical compositions of or containing NHP, or for raising antibodies to a NHP, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors (Pharmacia or American Type Culture Collection) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target expression product can be released from the GST moiety.

[0057] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign polynucleotide sequences. The virus grows in Spodoptera frugiperda cells. A NHP coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of NHP coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted sequence is expressed (e.g., see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0058] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the NHP nucleotide sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric sequence may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a NHP product in infected hosts (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted NHP nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire NHP gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of a NHP coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bitter et al., 1987, Methods in Enzymol. 153:516-544).

[0059] In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the expression product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and expression products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the expression product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, human cell lines.

[0060] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the NHP sequences described above can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines that express the NHP product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the NHP product.

[0061] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

[0062] Alternatively, any fusion protein can be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the sequence of interest is subcloned into a vaccinia recombination plasmid such that the sequence's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺-nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

[0063] Also encompassed by the present invention are fusion proteins that direct the NHP to a target organ and/or facilitate transport across the membrane into the cytosol. Conjugation of NHPs to antibody molecules or their Fab fragments could be used to target cells bearing a particular epitope. Attaching the appropriate signal sequence to the NHP would also transport the NHP to the desired location within the cell. Alternatively targeting of NHP or its nucleic acid sequence might be achieved using liposome or lipid complex based delivery systems. Such technologies are described in “Liposomes:A Practical Approach”, New, R. R. C., ed., Oxford University Press, New York and in U.S. Pat. Nos. 4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respective disclosures, which are herein incorporated by reference in their entirety. Additionally embodied are novel protein constructs engineered in such a way that they facilitate transport of the NHP to the target site or desired organ, where they cross the cell membrane and/or the nucleus where the NHP can exert its functional activity. This goal may be achieved by coupling of the NHP to a cytokine or other ligand that provides targeting specificity, and/or to a protein transducing domain (see generally U.S. applications Ser. Nos. 60/111,701 and 60/056,713, both of which are herein incorporated by reference, for examples of such transducing sequences) to facilitate passage across cellular membranes and can optionally be engineered to include nuclear localization.

5.3 ANTIBODIES TO NHP PRODUCTS

[0064] Antibodies that specifically recognize one or more epitopes of a NHP, or epitopes of conserved variants of a NHP, or peptide fragments of a NHP are also encompassed by the invention. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0065] The antibodies of the invention may be used, for example, in the detection of NHP in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal amounts of NHP. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes for the evaluation of the effect of test compounds on expression and/or activity of a NHP expression product. Additionally, such antibodies can be used in conjunction gene therapy to, for example, evaluate the normal and/or engineered NHP-expressing cells prior to their introduction into the patient. Such antibodies may additionally be used as a method for the inhibition of abnormal NHP activity. Thus, such antibodies may, therefore, be utilized as part of treatment methods.

[0066] For the production of antibodies, various host animals may be immunized by injection with a NHP, an NHP peptide (e.g., one corresponding to a functional domain of an NHP), truncated NHP polypeptides (NHP in which one or more domains have been deleted), functional equivalents of the NHP or mutated variant of the NHP. Such host animals may include but are not limited to pigs, rabbits, mice, goats, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's adjuvant (complete and incomplete), mineral salts such as aluminum hydroxide or aluminum phosphate, chitosan, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Alternatively, the immune response could be enhanced by combination and or coupling with molecules such as keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, cholera toxin or fragments thereof. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.

[0067] Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

[0068] In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Such technologies are described in U.S. Pat. Nos. 6,075,181 and 5,877,397 and their respective disclosures, which are herein incorporated by reference in their entirety. Also encompassed by the present invention is the use of fully humanized monoclonal antibodies as described in U.S. Pat. No. 6,150,584 and respective disclosures, which are herein incorporated by reference in their entirety.

[0069] Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adapted to produce single chain antibodies against NHP expression products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

[0070] Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such fragments include, but are not limited to: the F(ab′)₂ fragments, which can be produced by pepsin digestion of the antibody molecule and the Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

[0071] Antibodies to a NHP can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” a given NHP, using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). For example antibodies that bind to a NHP domain and competitively inhibit the binding of NHP to its cognate receptor can be used to generate anti-idiotypes that “mimic” the NHP and, therefore, bind and activate or neutralize a receptor. Such anti-idiotypic antibodies or Fab fragments of such anti-idiotypes can be used in therapeutic regimens involving a NHP mediated pathway.

[0072] Additionally given the high degree of relatedness of mammalian NHPs, the presently described knock-out mice (having never seen NHP, and thus never been tolerized to NHP) have a unique utility, as they can be advantageously applied to the generation of antibodies against the disclosed mammalian NHP (i.e., NHP will be immunogenic in NHP knock-out animals).

[0073] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. All cited publications, patents, and patent applications are herein incorporated by reference in their entirety.

1 7 1 1812 DNA homo sapiens 1 atggcgctcc gcgggaccct ccggccgctc aaagttcgca ggaggcgaga gatgctgccg 60 cagcaagtcg gcttcgtgtg cgcggtgctg gccctggtgt gctgtgcgtc cggcctcttc 120 ggcagcttgg ggcacaaaac agcttctgct agcaaacgtg tcctgccaga cacatggaga 180 aatagaaagt tgatggcccc agtgaatggg acacagacag ccaagaactg cacagatcct 240 gcgattcacg agttccccac agatctgttc tccaataagg agcgacagca cggagccgtc 300 ctgctgcaca tccttggtgc tctgtatatg ttctatgcct tggccatagt gtgcgatgac 360 ttctttgttc cgtctctaga gaagatctgt gagagactcc atctgagcga agatgtggct 420 ggagccacct tcatggctgc aggaagctca acgccagagc tgtttgcgtc tgttattggg 480 gtgttcatca cccaygggga cgtcggggtg ggcaccatcg tgggctctgc tgtgttcaac 540 atcctgtgca taattggagt gtgcggactg tttgctggcc aggtggtccg tctgacgtgg 600 tgggccgtgt gccgagactc cgtgtactac accatctctg tcatcgtgct catcgtgttc 660 atatatgatg aacaaattgt gtggtgggaa ggcctggtgc tcatcatctt gtatgtgttt 720 tatattctga tcatgaagta caatgtgaag atgcaagcct ttttcacagt caaacaaaag 780 agcattgcaa acggtaaccc ggtcaacagt gagctggagg ctgtgaagga gaagccacag 840 tatggcaaga accccgtggt gatggtggac gagattatga gctccagccc tcccaagttc 900 accttccctg aagcaggctt acgaatcatg atcaccaata agtttggacc caggacccga 960 ctacggatgg ccagcaggat catcattaat gagcggcaga gactgatcaa ctcggccaat 1020 ggtgtgagca gtaagccgct tcaaaacggg aggcacgaga acattgagaa cgggaatgtt 1080 cctgtggaaa accccgaaga ccctcagcag aatcaggagc agcagccgcc gccacagcca 1140 ccaccgccag agccagagcc ggtggaggct gacttcctgt cccccttctc cgtgccggag 1200 gccagagggg acaaggtcaa gtgggtgttc acctggcccc tcatcttcct cctgtgcgtc 1260 accattccca actgcagcaa gccccgctgg gagaagttct tcatggtcac cttcatcacc 1320 gccacgctgt ggatcgctgt gttctcctac atcatggtgt ggctggtgac tattatcgga 1380 tacacacttg ggatcccgga tgtcatcatg ggcattactt tcctggcagc agggacaagt 1440 gttccagact gcatggccag cctaattgtg gcgagacaag gccttgggga catggcagtc 1500 tccaacacca taggaagcaa cgtgtttgac atcctggtag gacttggtgt accgtggggc 1560 ctgcagacca tggttgttaa ttatggatca acagtgaaga tcaacagccg ggggctggtc 1620 tattccgtgg tcctgttgct gggctctgtc gctctcaccg tcctcggcat ccacctaaac 1680 aagtggcgac tggaccggaa gctgggtgtc tacgtgctgg ttctctacgc catcttcttg 1740 tgcttctcca taatgataga gtttaacgtc tttaccttcg tcaacttgcc gatgtgccgg 1800 gaagacgatt ag 1812 2 603 PRT homo sapiens 2 Met Ala Leu Arg Gly Thr Leu Arg Pro Leu Lys Val Arg Arg Arg Arg 1 5 10 15 Glu Met Leu Pro Gln Gln Val Gly Phe Val Cys Ala Val Leu Ala Leu 20 25 30 Val Cys Cys Ala Ser Gly Leu Phe Gly Ser Leu Gly His Lys Thr Ala 35 40 45 Ser Ala Ser Lys Arg Val Leu Pro Asp Thr Trp Arg Asn Arg Lys Leu 50 55 60 Met Ala Pro Val Asn Gly Thr Gln Thr Ala Lys Asn Cys Thr Asp Pro 65 70 75 80 Ala Ile His Glu Phe Pro Thr Asp Leu Phe Ser Asn Lys Glu Arg Gln 85 90 95 His Gly Ala Val Leu Leu His Ile Leu Gly Ala Leu Tyr Met Phe Tyr 100 105 110 Ala Leu Ala Ile Val Cys Asp Asp Phe Phe Val Pro Ser Leu Glu Lys 115 120 125 Ile Cys Glu Arg Leu His Leu Ser Glu Asp Val Ala Gly Ala Thr Phe 130 135 140 Met Ala Ala Gly Ser Ser Thr Pro Glu Leu Phe Ala Ser Val Ile Gly 145 150 155 160 Val Phe Ile Thr His Gly Asp Val Gly Val Gly Thr Ile Val Gly Ser 165 170 175 Ala Val Phe Asn Ile Leu Cys Ile Ile Gly Val Cys Gly Leu Phe Ala 180 185 190 Gly Gln Val Val Arg Leu Thr Trp Trp Ala Val Cys Arg Asp Ser Val 195 200 205 Tyr Tyr Thr Ile Ser Val Ile Val Leu Ile Val Phe Ile Tyr Asp Glu 210 215 220 Gln Ile Val Trp Trp Glu Gly Leu Val Leu Ile Ile Leu Tyr Val Phe 225 230 235 240 Tyr Ile Leu Ile Met Lys Tyr Asn Val Lys Met Gln Ala Phe Phe Thr 245 250 255 Val Lys Gln Lys Ser Ile Ala Asn Gly Asn Pro Val Asn Ser Glu Leu 260 265 270 Glu Ala Val Lys Glu Lys Pro Gln Tyr Gly Lys Asn Pro Val Val Met 275 280 285 Val Asp Glu Ile Met Ser Ser Ser Pro Pro Lys Phe Thr Phe Pro Glu 290 295 300 Ala Gly Leu Arg Ile Met Ile Thr Asn Lys Phe Gly Pro Arg Thr Arg 305 310 315 320 Leu Arg Met Ala Ser Arg Ile Ile Ile Asn Glu Arg Gln Arg Leu Ile 325 330 335 Asn Ser Ala Asn Gly Val Ser Ser Lys Pro Leu Gln Asn Gly Arg His 340 345 350 Glu Asn Ile Glu Asn Gly Asn Val Pro Val Glu Asn Pro Glu Asp Pro 355 360 365 Gln Gln Asn Gln Glu Gln Gln Pro Pro Pro Gln Pro Pro Pro Pro Glu 370 375 380 Pro Glu Pro Val Glu Ala Asp Phe Leu Ser Pro Phe Ser Val Pro Glu 385 390 395 400 Ala Arg Gly Asp Lys Val Lys Trp Val Phe Thr Trp Pro Leu Ile Phe 405 410 415 Leu Leu Cys Val Thr Ile Pro Asn Cys Ser Lys Pro Arg Trp Glu Lys 420 425 430 Phe Phe Met Val Thr Phe Ile Thr Ala Thr Leu Trp Ile Ala Val Phe 435 440 445 Ser Tyr Ile Met Val Trp Leu Val Thr Ile Ile Gly Tyr Thr Leu Gly 450 455 460 Ile Pro Asp Val Ile Met Gly Ile Thr Phe Leu Ala Ala Gly Thr Ser 465 470 475 480 Val Pro Asp Cys Met Ala Ser Leu Ile Val Ala Arg Gln Gly Leu Gly 485 490 495 Asp Met Ala Val Ser Asn Thr Ile Gly Ser Asn Val Phe Asp Ile Leu 500 505 510 Val Gly Leu Gly Val Pro Trp Gly Leu Gln Thr Met Val Val Asn Tyr 515 520 525 Gly Ser Thr Val Lys Ile Asn Ser Arg Gly Leu Val Tyr Ser Val Val 530 535 540 Leu Leu Leu Gly Ser Val Ala Leu Thr Val Leu Gly Ile His Leu Asn 545 550 555 560 Lys Trp Arg Leu Asp Arg Lys Leu Gly Val Tyr Val Leu Val Leu Tyr 565 570 575 Ala Ile Phe Leu Cys Phe Ser Ile Met Ile Glu Phe Asn Val Phe Thr 580 585 590 Phe Val Asn Leu Pro Met Cys Arg Glu Asp Asp 595 600 3 951 DNA homo sapiens 3 atggtggacg agattatgag ctccagccct cccaagttca ccttccctga agcaggctta 60 cgaatcatga tcaccaataa gtttggacct aggacccgac tacggatggc cagcaggatc 120 atcattaatg agcggcagag actgatcaac tcggccaatg gtgtgagcag taagccgctt 180 caaaacggga ggcacgagaa cattgagaac gggaatgttc ctgtggaaaa ccccgaagac 240 cctcagcaga atcaggagca gcagccgccg ccacagccac caccgccaga gccagagccg 300 gtggaggctg acttcctgtc ccccttctcc gtgccggagg ccagagggga caaggtcaag 360 tgggtgttca cctggcccct catcttcctc ctgtgcgtca ccattcccaa ctgcagcaag 420 ccccgctggg agaagttctt catggtcacc ttcatcaccg ccacgctgtg gatcgctgtg 480 ttctcctaca tcatggtgtg gctggtgact attatcggat acacacttgg gatcccggat 540 gtcatcatgg gcattacttt cctggcagca gggacaagtg ttccagactg catggccagc 600 ctaattgtgg cgagacaagg ccttggggac atggcagtct ccaacaccat aggaagcaac 660 gtgtttgaca tcctggtagg acttggtgta ccgtggggcc tgcagaccat ggttgttaat 720 tatggatcaa cagtgaagat caacagccgg gggctggtct attccgtggt cctgttgctg 780 ggctctgtcg ctctcaccgt cctcggcatc cacctaaaca agtggcgact ggaccggaag 840 ctgggtgtct acgtgctggt tctctacgcc atcttcttgt gcttctccat aatgatagag 900 tttaacgtct ttaccttcgt caacttgccg atgtgccggg aagacgatta g 951 4 316 PRT homo sapiens 4 Met Val Asp Glu Ile Met Ser Ser Ser Pro Pro Lys Phe Thr Phe Pro 1 5 10 15 Glu Ala Gly Leu Arg Ile Met Ile Thr Asn Lys Phe Gly Pro Arg Thr 20 25 30 Arg Leu Arg Met Ala Ser Arg Ile Ile Ile Asn Glu Arg Gln Arg Leu 35 40 45 Ile Asn Ser Ala Asn Gly Val Ser Ser Lys Pro Leu Gln Asn Gly Arg 50 55 60 His Glu Asn Ile Glu Asn Gly Asn Val Pro Val Glu Asn Pro Glu Asp 65 70 75 80 Pro Gln Gln Asn Gln Glu Gln Gln Pro Pro Pro Gln Pro Pro Pro Pro 85 90 95 Glu Pro Glu Pro Val Glu Ala Asp Phe Leu Ser Pro Phe Ser Val Pro 100 105 110 Glu Ala Arg Gly Asp Lys Val Lys Trp Val Phe Thr Trp Pro Leu Ile 115 120 125 Phe Leu Leu Cys Val Thr Ile Pro Asn Cys Ser Lys Pro Arg Trp Glu 130 135 140 Lys Phe Phe Met Val Thr Phe Ile Thr Ala Thr Leu Trp Ile Ala Val 145 150 155 160 Phe Ser Tyr Ile Met Val Trp Leu Val Thr Ile Ile Gly Tyr Thr Leu 165 170 175 Gly Ile Pro Asp Val Ile Met Gly Ile Thr Phe Leu Ala Ala Gly Thr 180 185 190 Ser Val Pro Asp Cys Met Ala Ser Leu Ile Val Ala Arg Gln Gly Leu 195 200 205 Gly Asp Met Ala Val Ser Asn Thr Ile Gly Ser Asn Val Phe Asp Ile 210 215 220 Leu Val Gly Leu Gly Val Pro Trp Gly Leu Gln Thr Met Val Val Asn 225 230 235 240 Tyr Gly Ser Thr Val Lys Ile Asn Ser Arg Gly Leu Val Tyr Ser Val 245 250 255 Val Leu Leu Leu Gly Ser Val Ala Leu Thr Val Leu Gly Ile His Leu 260 265 270 Asn Lys Trp Arg Leu Asp Arg Lys Leu Gly Val Tyr Val Leu Val Leu 275 280 285 Tyr Ala Ile Phe Leu Cys Phe Ser Ile Met Ile Glu Phe Asn Val Phe 290 295 300 Thr Phe Val Asn Leu Pro Met Cys Arg Glu Asp Asp 305 310 315 5 1062 DNA homo sapiens 5 atgcaagcct ttttcacagt caaacaaaag agcattgcaa acggtaaccc ggtcaacagt 60 gagctggagg ctgtgaagga gaagccacag tatggcaaga accccgtggt gatggtggac 120 gagattatga gctccagccc tcccaagttc accttccctg aagcaggctt acgaatcatg 180 atcaccaata agtttggacc caggacccga ctacggatgg ccagcaggat catcattaat 240 gagcggcaga gactgatcaa ctcggccaat ggtgtgagca gtaagccgct tcaaaacggg 300 aggcacgaga acattgagaa cgggaatgtt cctgtggaaa accccgaaga ccctcagcag 360 aatcaggagc agcagccgcc gccacagcca ccaccgccag agccagagcc ggtggaggct 420 gacttcctgt cccccttctc cgtgccggag gccagagggg acaaggtcaa gtgggtgttc 480 acctggcccc tcatcttcct cctgtgcgtc accattccca actgcagcaa gccccgctgg 540 gagaagttct tcatggtcac cttcatcacc gccacgctgt ggatcgctgt gttctcctac 600 atcatggtgt ggctggtgac tattatcgga tacacacttg ggatcccgga tgtcatcatg 660 ggcattactt tcctggcagc agggacaagt gttccagact gcatggccag cctaattgtg 720 gcgagacaag gccttgggga catggcagtc tccaacacca taggaagcaa cgtgtttgac 780 atcctggtag gacttggtgt accgtggggc ctgcagacca tggttgttaa ttatggatca 840 acagtgaaga tcaacagccg ggggctggtc tattccgtgg tcctgttgct gggctctgtc 900 gctctcaccg tcctcggcat ccacctaaac aagtggcgac tggaccggaa gctgggtgtc 960 tacgtgctgg ttctctacgc catcttcttg tgcttctcca taatgataga gtttaacgtc 1020 tttaccttcg tcaacttgcc gatgtgccgg gaagacgatt ag 1062 6 353 PRT homo sapiens 6 Met Gln Ala Phe Phe Thr Val Lys Gln Lys Ser Ile Ala Asn Gly Asn 1 5 10 15 Pro Val Asn Ser Glu Leu Glu Ala Val Lys Glu Lys Pro Gln Tyr Gly 20 25 30 Lys Asn Pro Val Val Met Val Asp Glu Ile Met Ser Ser Ser Pro Pro 35 40 45 Lys Phe Thr Phe Pro Glu Ala Gly Leu Arg Ile Met Ile Thr Asn Lys 50 55 60 Phe Gly Pro Arg Thr Arg Leu Arg Met Ala Ser Arg Ile Ile Ile Asn 65 70 75 80 Glu Arg Gln Arg Leu Ile Asn Ser Ala Asn Gly Val Ser Ser Lys Pro 85 90 95 Leu Gln Asn Gly Arg His Glu Asn Ile Glu Asn Gly Asn Val Pro Val 100 105 110 Glu Asn Pro Glu Asp Pro Gln Gln Asn Gln Glu Gln Gln Pro Pro Pro 115 120 125 Gln Pro Pro Pro Pro Glu Pro Glu Pro Val Glu Ala Asp Phe Leu Ser 130 135 140 Pro Phe Ser Val Pro Glu Ala Arg Gly Asp Lys Val Lys Trp Val Phe 145 150 155 160 Thr Trp Pro Leu Ile Phe Leu Leu Cys Val Thr Ile Pro Asn Cys Ser 165 170 175 Lys Pro Arg Trp Glu Lys Phe Phe Met Val Thr Phe Ile Thr Ala Thr 180 185 190 Leu Trp Ile Ala Val Phe Ser Tyr Ile Met Val Trp Leu Val Thr Ile 195 200 205 Ile Gly Tyr Thr Leu Gly Ile Pro Asp Val Ile Met Gly Ile Thr Phe 210 215 220 Leu Ala Ala Gly Thr Ser Val Pro Asp Cys Met Ala Ser Leu Ile Val 225 230 235 240 Ala Arg Gln Gly Leu Gly Asp Met Ala Val Ser Asn Thr Ile Gly Ser 245 250 255 Asn Val Phe Asp Ile Leu Val Gly Leu Gly Val Pro Trp Gly Leu Gln 260 265 270 Thr Met Val Val Asn Tyr Gly Ser Thr Val Lys Ile Asn Ser Arg Gly 275 280 285 Leu Val Tyr Ser Val Val Leu Leu Leu Gly Ser Val Ala Leu Thr Val 290 295 300 Leu Gly Ile His Leu Asn Lys Trp Arg Leu Asp Arg Lys Leu Gly Val 305 310 315 320 Tyr Val Leu Val Leu Tyr Ala Ile Phe Leu Cys Phe Ser Ile Met Ile 325 330 335 Glu Phe Asn Val Phe Thr Phe Val Asn Leu Pro Met Cys Arg Glu Asp 340 345 350 Asp 7 2366 DNA homo sapiens 7 ccgacctcgc cctcgggcca tgaggctttg gcccggagct cctcgcctct gagtcgcgca 60 ccgcctgctc cagccccagc gccgctcggc cactgattgc actctggccg ctgaagctcc 120 ccatcctctc ccagagacgg cacccaggcg ctccgggatg gcgctccgcg ggaccctccg 180 gccgctcaaa gttcgcagga ggcgagagat gctgccgcag caagtcggct tcgtgtgcgc 240 ggtgctggcc ctggtgtgct gtgcgtccgg cctcttcggc agcttggggc acaaaacagc 300 ttctgctagc aaacgtgtcc tgccagacac atggagaaat agaaagttga tggccccagt 360 gaatgggaca cagacagcca agaactgcac agatcctgcg attcacgagt tccccacaga 420 tctgttctcc aataaggagc gacagcacgg agccgtcctg ctgcacatcc ttggtgctct 480 gtatatgttc tatgccttgg ccatagtgtg cgatgacttc tttgttccgt ctctagagaa 540 gatctgtgag agactccatc tgagcgaaga tgtggctgga gccaccttca tggctgcagg 600 aagctcaacg ccagagctgt ttgcgtctgt tattggggtg ttcatcaccc ayggggacgt 660 cggggtgggc accatcgtgg gctctgctgt gttcaacatc ctgtgcataa ttggagtgtg 720 cggactgttt gctggccagg tggtccgtct gacgtggtgg gccgtgtgcc gagactccgt 780 gtactacacc atctctgtca tcgtgctcat cgtgttcata tatgatgaac aaattgtgtg 840 gtgggaaggc ctggtgctca tcatcttgta tgtgttttat attctgatca tgaagtacaa 900 tgtgaagatg caagcctttt tcacagtcaa acaaaagagc attgcaaacg gtaacccggt 960 caacagtgag ctggaggctg tgaaggagaa gccacagtat ggcaagaacc ccgtggtgat 1020 ggtggacgag attatgagct ccagccctcc caagttcacc ttccctgaag caggcttacg 1080 aatcatgatc accaataagt ttggacccag gacccgacta cggatggcca gcaggatcat 1140 cattaatgag cggcagagac tgatcaactc ggccaatggt gtgagcagta agccgcttca 1200 aaacgggagg cacgagaaca ttgagaacgg gaatgttcct gtggaaaacc ccgaagaccc 1260 tcagcagaat caggagcagc agccgccgcc acagccacca ccgccagagc cagagccggt 1320 ggaggctgac ttcctgtccc ccttctccgt gccggaggcc agaggggaca aggtcaagtg 1380 ggtgttcacc tggcccctca tcttcctcct gtgcgtcacc attcccaact gcagcaagcc 1440 ccgctgggag aagttcttca tggtcacctt catcaccgcc acgctgtgga tcgctgtgtt 1500 ctcctacatc atggtgtggc tggtgactat tatcggatac acacttggga tcccggatgt 1560 catcatgggc attactttcc tggcagcagg gacaagtgtt ccagactgca tggccagcct 1620 aattgtggcg agacaaggcc ttggggacat ggcagtctcc aacaccatag gaagcaacgt 1680 gtttgacatc ctggtaggac ttggtgtacc gtggggcctg cagaccatgg ttgttaatta 1740 tggatcaaca gtgaagatca acagccgggg gctggtctat tccgtggtcc tgttgctggg 1800 ctctgtcgct ctcaccgtcc tcggcatcca cctaaacaag tggcgactgg accggaagct 1860 gggtgtctac gtgctggttc tctacgccat cttcttgtgc ttctccataa tgatagagtt 1920 taacgtcttt accttcgtca acttgccgat gtgccgggaa gacgattagc gctgagtcgc 1980 ggcccctggg agctgatctg gacaccctgt gacactggcg tcctcctctc ccctccttcc 2040 cccaccacag gtctctcctg cataggcagc cactgtccgt tctttcacac actggaagga 2100 agagccatcg tggtctttgt ctggccacag gccaggctgc tgggcatcct cctcctcctt 2160 ggagttccac ccctgcaagg ctggatttgg gggccattat ctgagcagct tcaaagaccc 2220 ctgagctgcc aaccacggag atgtgccaag catctcatct ctcctgcaca ctttagtcag 2280 aaggacttct gcatgcagtt tgtctttctg ttctgcaggc agcttcagaa ttgaggtcat 2340 ttgtgagcac aagatctcat agggca 2366 

What is claimed is:
 1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence drawn from the group consisting of SEQ ID NOS: 2, 4, and
 6. 2. An isolated nucleic acid molecule comprising a nucleotide sequence that: (a) encodes the amino acid sequence shown in SEQ ID NO: 2; and (b) hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO:1 or the complement thereof.
 3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2.
 4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:4.
 5. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:6. 